1a. Objectives (from AD-416):
To identify genes contributing to resistance that may be useful in transgenic approaches and begin to characterize Hessian fly-responsive wheat gene promoters. Identify insecticidal proteins from non-wheat sources that may be useful against Hessian fly and develop germplasm for use in genomics/proteomics experiments.

1b. Approach (from AD-416):
We already have a large time-course microarray and a laser-capture micro-dissection microarray that have been analyzed but not completely mined for location-specific differences and compatible versus incompatible interaction differences. We will perform RNA-Seq on Hamlet wheat plants that generate a hypersensitive response, giving a different type of resistance from previous experiments for comparison of incompatible and compatible interactions. We will generate RNA-Seq data for Brachypodium response to Hf, which gives an intermediate interaction: plants are stunted some but still make seed, larvae develop farther than on wheat but do not emerge as adults. Through bioinformatics analyses, we will compare expression profiles among different data sets to identify genes specific to interaction type (compatible vs. incompatible) response type (standard versus hypersensitive/oxidative burst-based resistance), tissue location (global versus feeding site-specific) and host type (wheat versus Brachypodium) to yield a more complete view of resistance and new genes that were not detected in early experiments. In addition, these results will give us raw material for selecting genes for promoter studies.
Feeding assays utilizing our new technique (Hessian fly in situ translocation assay) to deliver compounds to the larvae via the plant, will use endogenous wheat antinutrient proteins and proteins from unrelated organisms to determine whether these proteins contribute to resistance by acting as acute toxins, disrupting/binding larval midgut, inducing behavioral changes in larvae or limiting their growth.
We have already cloned Hessian fly-responsive wheat promoters for Hfr-1, Wci-1, Dir, Wci-2 and Hfr-3. We are considering 20 additional genes and will target those with low expression in the seed under many different stress conditions. A collaborator is producing T1 plants with Wci-1, Hfr-3 and Wci-2 promoters driving GUS for future analysis of positional expression in response to Hessian fly attack. We will clone some Hessian fly-responsive promoters from genes discovered in other microarrays and RNA Seq experiments. With collaborators willing to transform wheat, we will hook endogenous wheat antinutrient genes to promising promoters to test the efficacy of the constructs as a new type of resistance. We will analyze promoter sequences to identify motifs common to Hessian fly-responsive genes and elements characterized in other plant genes.

3. Progress Report:
Hessian fly-responsive wheat gene promoters are needed to drive engineered genes that will provide new modes of resistance against the insect pest. We characterized the expression of several Hessian fly-responsive wheat genes and pursued a promising promoter for cloning. In order to be useful in the development of resistant germplasm, the promoter must respond quickly, within a few hours of attack, be expressed in vegetative tissue at the insect feeding site and not be expressed in the wheat seed, which will be consumed as a human food source. The promoter that we cloned meets the above criteria. Over 1000 base pairs of the promoter were cloned and sequenced. The clone was then sent to a collaborator who will use it to drive a reporter gene so that we will learn the site(s) of expression and level of response. This will help determine the potential of this promoter for use in engineering resistant plants.
Wheat genes encoding transcription factors act as master switches, often inducing a suite of genes for coordinate expression to make a plant either resistant or susceptible to insect pests and pathogens. We identified several Hessian fly-induced wheat transcription factor genes through a microarray experiment. One transcription factor gene is responsive only in susceptible plants and is a candidate for gene silencing in order to block the ability of a wheat plant to become susceptible. This gene and its promoter are being cloned for further study and future gene-silencing studies.
Through an in-planta feeding assay several Bt cry toxins were tested for their efficacy in killing or inhibiting the growth and development of first-instar Hessian fly larvae. One cry toxin was quite effective in slowing the development of larvae. The gene that encodes the cry toxin is a good candidate for transgenic Hessian fly resistance, especially if coupled with a wheat promoter that responds quickly to Hessian fly attack.
An understanding of the expression of Hessian fly genes that promote wheat susceptibility and allow the insect to feed on its host is important for developing strategies for targeting larval processes in transgenic resistance. We have characterized and quantified the expression of several genes in the Hessian fly polyamine and amino acid biosynthesis pathways, as well as the tricarboxylic acid cycle and glycolysis. Wheat genes that disrupt these processes will be pursued.
RNA-Seq experiments were conducted in order to identify Hessian fly genes that could be targeted for manipulation or silencing. The data files are currently under analysis.